JP5867890B1 - Virus-like particles used in immunoassay, blocking agents used therefor, and kits containing these - Google Patents

Abstract

An object of the present invention is to provide an immunological measurement method which is excellent in detection sensitivity and significantly suppresses detection background. The solution is a virus-like particle, wherein the virus-like particle contains a protein having a self-organization ability, and the physiological activity is achieved through a thiol group of at least one cysteine residue of the protein having the self-assembly ability. It is to provide virus-like particles that are modified by molecules.

Description

  The present invention relates to virus-like particles used in immunological assays, blocking agents used therefor, and kits containing these.

  Methods using various particles as immunoassay sensors are known (Patent Documents 1 to 8). For example, Patent Document 1 or Patent Document 2 proposes a method of using fluorescent semiconductor nanoparticles or silica particles as a detection element for immunoassay, which is a technique of performing immunoassay with fluorescence generated by nanoparticles.

  On the other hand, Patent Document 8 presents a nanoparticle conjugate in which various peptides are bound using a metal material, a magnetic material, or a semiconductor material as a nucleus.

  A virus-like particle having a hepatitis B virus surface antigen, wherein the binding domain (Z tag) for the Fc region of a protein A-derived antibody is tandem in the Pre-S1 and Pre-S2 regions at the N-terminus of such antigen. 2 (hereinafter sometimes referred to as ZZ-BNC) are pre-S1 and Pre- of L-type hepatitis B virus particles (patent document 9) produced by genetic recombination using yeast. Two binding domains (Z tags) for the Fc region of an antibody derived from protein A are inserted into the S2 region, and its usefulness as a production method and a drug delivery system is described in Patent Document 10.

  Paying attention to the function of this BNC-ZZ as a high sensor element, Patent Document 3 discloses a method of using an enzyme-labeled antibody and BNC-ZZ in combination, a method of detecting an antibody using fluorescently labeled BNC-ZZ, and a method of using an immunoassay. It is illustrated as an example. Patent Document 6 exemplifies a technique for increasing sensitivity by using BNC-ZZ for alignment of immobilized antibodies. Patent Document 4 describes a method of using biotinylated BNC-ZZ for immunological measurement. By binding biotinylated HRP enzyme or biotinylated antibody to biotinylated BNC-ZZ via streptavidin. It is exemplified in the Examples that the sensitivity can be increased more than using BNC-ZZ that is not biotinylated.

  In Patent Document 5, a method for producing a hybrid type particle having a protein molecule having a ZZ tag and a protein molecule not having a Pre-S region in order to increase the sensitivity of immunological measurement by increasing the antibody binding ability of BNC-ZZ particles. And how to use it. Further, in Patent Document 7, a method for simultaneously detecting multiple antigens by weakly crosslinking an antibody to fluorescently labeled BNC-ZZ is exemplified in the Examples.

JP-T-2006-517985 Special Table 2002-544488 JP 2007-127626 A JP 2008-191143 A JP 2010-096677 A JP 2007-121276 A JP 2010-210444 A Special table 2007-506084 gazette JP 2001-316298 A JP 2004-002313 A

Vaccine 19, 3154-3163, 2001

  In general, in order to increase sensitivity in an immunoassay, (1) an element having a high affinity for the detection target substance of the detection element is used, and (2) the signal intensity generated by the detection element bound to the detection target substance is increased. Any method of raising is used. Here, when the above-described BNC-ZZ is used as a detection element, the affinity to the detection target is constant, and therefore, the most effective means is to increase the generated signal intensity.

  However, in a document that discloses the use of BNC-ZZ, a method for increasing the signal by increasing the amount of HRP binding using the well-known specific binding ability of biotin and streptavidin is known as biotinylated BNC. It is only used as -ZZ (Patent Document 4) and has not been studied.

Since ZZ-BNC has a protein A-derived antibody binding site, binding to antibodies such as mouse IgG ₁ , rat IgG, sheep IgG ₁ , goat IgG ₁ and human IgG _{3 that} are important in immunoassay is weak. Usage and range of use are greatly limited. Furthermore, since ZZ-BNC binds to various IgGs, binding to antibodies other than the target occurs in the environment where multiple antibodies exist, such as antibody sandwich ELISA or serum containing multiple antibodies. It is difficult to detect.

  And when practical application is considered, since BNC-ZZ, its labeled body, and a modified body are lipoproteins, they show non-specific binding to glass and plastic. Non-specific adsorption not only greatly affects the immunoassay, but also becomes a serious problem even when the diluted solution is stored for a long time.

  As a result of intensive studies to solve the above problems, the present inventors have labeled such a virus-like particle when a bioactive molecule is labeled through a specific site of a protein having a self-organizing ability contained in the virus-like particle. It has been found that it can be suitably used for immunological measurement.

  Furthermore, it discovered that the specific blocking agent exhibited the outstanding blocking effect, when using the above-mentioned virus-like particle | grain for immunological measurement.

  The present invention has been completed based on such findings, and includes the inventions of the following broad aspects.

  Item 1. A virus-like particle for immunological measurement comprising a protein having self-assembly ability, which is modified by a bioactive molecule via a thiol group of at least one cysteine residue of the protein having self-assembly ability. A virus-like particle.

  Item 2. The virus-like particle according to Item 1, wherein the protein having the ability to self-assemble is an HBsAg protein.

  Item 3. The virus-like particle according to Item 1, wherein the protein having the ability to self-assemble comprises the amino acid sequence shown in SEQ ID NO: 1.

  Item 4. The virus-like particle according to any one of Items 1 to 3, wherein the protein having the ability to self-assemble has an antibody binding domain.

  Item 5. The bioactive molecule according to any one of Items 1 to 4, wherein the physiologically active molecule is at least one selected from the group consisting of an enzyme, an antibody binding domain, biotin, a fluorescent dye, a luminescent dye, and an avidin compound. The virus-like particle described.

  Item 6 The virus-like particle according to Item 5, wherein the enzyme is alkaline phosphatase and / or peroxidase.

  Item 7 The above-mentioned antibody binding domain is at least one selected from the group consisting of an antibody binding domain contained in protein A, an antibody binding domain contained in protein G, and an antibody binding domain contained in protein L. 6. The virus-like particle according to item 4 or item 5.

  Item 8 The virus particle according to Item 4 or Item 5, wherein the antibody-binding domain has an amino acid sequence represented by any one of SEQ ID NOs: 3 to 5.

  Item 9 The item 5 above, wherein the avidin compound is at least one selected from the group consisting of avidin, streptavidin, neutravidin, AVR protein, bradavidin, rhizavidin, and tamavidin (registered trademark). The virus-like particle according to 1.

  Item 10 The virus-like particle according to any one of Items 1 to 9, wherein the bioactive molecule is an antibody-binding domain, and an antibody binds to the antibody-binding domain.

  Item 11 A blocking agent for immunological measurement using the virus-like particle according to any one of Items 1 to 10, wherein the blocking agent is hydroxyalkyl cellulose, polyvinyl alcohol, ethylene oxide / propylene oxide A blocking agent comprising at least one selected from the group consisting of a copolymer and a copolymer composed of 2-methacryloyloxyethylphosphocholine.

  Item 12. The blocking agent according to Item 11, wherein the hydroxyalkylcellulose is hydroxypropylmethylcellulose.

  Item 13 The blocking agent according to Item 11, wherein the degree of polymerization of the polyvinyl alcohol is 200 to 5,000.

  Item 14. The blocking agent according to Item 11, wherein the ethylene oxide / propylene oxide copolymer is Pluronic (registered trademark).

  Item 15. The blocking agent according to Item 11, wherein the ethylene oxide / propylene oxide copolymer is Pluronic (registered trademark) F127 and / or Pluronic (registered trademark) P105.

  Item 16. The blocking agent according to Item 11, wherein the copolymer composed of 2-methacryloyloxyethylphosphocholine is Biolipid (registered trademark).

  Item 17. The blocking agent according to Item 11, wherein the polymer having 2-methacryloyloxyethylphosphocholine as a structural unit is Biolipid (registered trademark) 206 and / or Biolipid (registered trademark) 802.

  Item 18 An immunological measurement kit comprising the virus-like particle according to any one of Items 1 to 10 and the blocking agent according to any one of Items 11 to 17.

  The virus-like particle according to the present invention can be immunologically measured with excellent detection sensitivity.

  The blocking agent according to the present invention exhibits an effect of lowering the background of the obtained data and / or sensitizing the detection signal in immunological measurement performed using the virus-like particle according to the present invention. The ratio can be increased significantly.

  According to the immunological measurement using the kit according to the present invention, the S / N ratio of the obtained data can be remarkably increased.

FIG. 6 shows the results of Example 5. The figure which shows the result of Example 6. FIG. The figure which shows the result of Example 16. The figure which shows the result of Example 17. The figure which shows the result of Example 18. FIG. The figure which shows the result of Example 20. FIG. The figure which shows the result of Example 21. FIG. The figure which shows the result of Example 22. The figure which shows the result of Example 23. FIG. The figure which shows the result of Example 24 (anti-GFP antibody). The figure which shows the result of Example 24 (HMG monoclonal antibody). The figure which shows the result of Example 25. FIG. The figure which shows the result of Example 27. FIG. The figure which shows the result of Example 28. FIG. The figure which shows the result of Example 29. The figure which shows the result of Example 30. The figure which shows the result of Example 31. The figure which shows the result of Example 32. FIG. The figure which shows the result of Example 33. FIG. The figure which shows the result of Example 34. The figure which shows the result of Example 35. The figure which shows the result of Example 36.

<Virus-like particles>
The virus-like particle according to the present invention is used in an immunological assay and contains a protein having a self-organizing ability. A protein having self-organization ability is obtained by modifying a bioactive molecule via a thiol group of a cysteine residue.

  A protein having a self-organizing ability is a protein that can form a virus-like particle by involving a lipid bilayer such as an endoplasmic reticulum lumen, a cell membrane, or a nuclear membrane in a living body, particularly a cell, and a cysteine residue. Although it does not specifically limit in the range to have, For example, the protein relevant to the budding function of the virus which has an envelope, an envelope protein, or these variants etc. are mentioned.

  Although the virus which has an envelope is not specifically limited, For example, the virus which belongs to Hepadnaviridae, such as hepatitis B virus (HBV) and duck hepatitis B virus; It belongs to Paramyxoviridae, such as Sendai virus (HVJ) Viruses; viruses belonging to the herpesviridae family such as herpes simplex virus; viruses belonging to the orthomyxoviridae family such as influenza virus; viruses belonging to the retroviridae family such as human immunodeficiency virus.

  The protein having the ability to self-assemble is not particularly limited. For example, hepatitis B virus surface antigen (HBsAg) protein that is a protein related to the budding function of HVB; F protein that is a protein related to the budding function of HVJ; Examples include hemagglutinin-neuraminidase protein, or variants thereof. Of these, HBsAg protein, F protein, hemagglutinin-neuraminidase protein, and variants thereof are preferred.

  The mutant is not particularly limited as long as it has at least one cysteine residue and exhibits an action capable of forming a virus-like particle as described above. The number of mutagenesis is not particularly limited within the same range, but usually 85% or more, preferably 90% or more, more preferably 95% or more, and most preferably 99% or more identity with the amino acid sequence before mutagenesis. The number of mutation introductions may be such that a mutant having

  The term “mutation introduction” as used herein includes substitution, deletion, insertion and the like. Specific mutagenesis can be carried out using a known method and is not particularly limited. For example, a conservative substitution technique may be employed for substitution. The term “conservative substitution technique” means that an amino acid residue is replaced with an amino acid residue having a similar side chain.

  For example, substitution with amino acid residues having basic side chains such as lysine, arginine, histidine and the like is a conservative substitution technique. Other amino acid residues having acidic side chains such as aspartic acid and glutamic acid; amino acid residues having uncharged polar side chains such as glycine, asparagine, glutamine, serine, threonine, tyrosine and cysteine; alanine, valine, leucine, Amino acid residues having non-polar side chains such as isoleucine, proline, phenylalanine, methionine, tryptophan, etc .; Amino acid residues having β-branched side chains such as threonine, valine, isoleucine, etc., fragrances such as tyrosine, phenylalanine, tryptophan, histidine, etc. Similarly, substitutions between amino acid residues having a family side chain are conservative substitutions.

  The term “identity” refers to the degree to which two or more comparable amino acid sequences are identical to each other. Therefore, the higher the identity of two amino acid sequences, the higher the identity or similarity of those sequences. The amino acid sequence or level of identity is determined, for example, using FASTA, a sequence analysis tool, using default parameters. Specific methods of these analysis methods are known, and the National Center of Biotechnology Information (NCBI) website (http://www.ncbi.nlm.nih.gov/) may be referred to.

  Although it does not specifically limit with HBsAg, For example, the protein etc. which contain the amino acid sequence shown to sequence number 1 are mentioned, For example, the variant of HBsAg is the variant of HBsAg etc. which are described in patent documents 5 and 9, etc. Can be mentioned. Although it does not specifically limit with F protein, For example, the protein etc. which consist of an amino acid sequence shown by Accession No. NP_056877; Version: NP_056877.1GI: 9627226 registered on the NCBI website are mentioned. The hemagglutinin-neuraminidase protein is not particularly limited, and examples thereof include a protein having an amino acid sequence represented by Accession No. NP — 056878; Version: NP — 056878.1 GI: 9627227 registered on the NCBI website.

  A protein having a self-organizing ability has a cysteine residue, and a bioactive molecule is modified through a thiol group of the cysteine residue. The specific cysteine residue is not particularly limited, but a bioactive molecule is modified with a specific cysteine residue located on the surface of a virus-like particle formed based on a protein having self-assembly ability. Is preferred.

  As such a cysteine residue, for example, in the case of the HBsAg protein consisting of the amino acid sequence shown in SEQ ID NO: 1, the 107th, 121st, 124th, 137th, 138th, 139th, 147th, or 149th position Of cysteine residues. In addition, the 9-28th, 80-98th, and 170-192nd amino acid sequences are estimated as a transmembrane domain of this HBsAg protein.

  For example, if it is F protein which consists of an amino acid sequence described in the said NCBI website, it is 70th, 199th, 338th, 347th, 362th, 370th, 394th, 399th, 401st, or 424 The second cysteine residue. In addition, as the transmembrane domain of this F protein, amino acid sequences of 1st to 25th, 117th to 139th, and 501st to 523rd are estimated.

  For example, if it is a hemagglutinin-neuraminidase protein consisting of the amino acid sequence described on the NCBI website, the 129th, 138th, 161st, 192nd, 216th, 258th, 271st, 352th, 357th, Examples include 365, 463, 469, 473, 535, 544, or 571 cysteine residues. In addition, the 38-60th amino acid sequence is estimated as a transmembrane domain of this hemagglutinin-neuraminidase protein.

  The physiologically active molecule is not particularly limited, and examples thereof include enzymes, antibody binding domains, biotin, fluorescent dyes, luminescent dyes, and avidin compounds. As described above, the protein according to the present invention has a plurality of cysteine residues, and a physiologically active molecule is bound via at least one cysteine residue in the protein. Among antibody binding domains, biotin, fluorescent dyes, luminescent dyes, avidin compounds and the like, not only one kind but also two or more kinds may be combined and bound via separate cysteine residues.

  The specific enzyme is not particularly limited as long as it is an enzyme in the field of immunological measurement, and examples thereof include peroxidase and alkaline phosphatase.

  The specific antibody binding domain is not particularly limited, but is preferably a domain that binds to the Fc domain of an antibody. Specific antibody binding domain is not particularly limited, but for example, antibody binding domain (SEQ ID NO: 3) contained in protein A, antibody binding domain (SEQ ID NO: 4) contained in protein G, and antibody contained in protein L Examples include a binding domain (SEQ ID NO: 5).

  The antibody binding domain included in the bioactive molecule may be an embodiment in which a plurality of such antibody binding domains are the same or different. As such an embodiment, for example, an antibody comprising an antibody binding domain contained in protein A, an antibody binding domain contained in protein G, and an antibody binding domain contained in protein G in that order from the N-terminal. Examples include a binding domain (SEQ ID NO: 6), a ZZ tag containing the antibody binding domain contained in protein A in tandem in order from the N-terminus.

  The antibody that binds to the antibody binding domain is not particularly limited, and the structure thereof is not limited to immunoglobulin, and any molecule having a structure that at least functions to recognize an antigen may be used. An antibody having a building block structure such as

  The origin of the antibody is not particularly limited, and antibodies derived from various animals suitable for antibody production can be used.

  For example, when the antibody is an immunoglobulin, its subtype is not particularly limited. Further, the subclass when the immunoglobulin is IgG, IgA or the like is not particularly limited.

When the above-mentioned bioactive molecule is an antibody-binding domain, an embodiment in which such a domain is bound to the above-described antibody is also included in the virus-like particle according to the present invention. In addition, the binding between the antibody binding domain and the antibody may be a strong binding (sometimes referred to as an irreversible binding in the present specification) using a known crosslinking agent. Known crosslinking agents include BS _{3 and the} like.

  Biotin, fluorescent dye, and luminescent dye are not particularly limited as long as they are usually used in the field of immunological measurement, and known ones may be used as appropriate.

  Specific avidin compounds are not particularly limited, and examples thereof include avidin, streptavidin, neutravidin, AVR protein, bradavidin, rhizavidin, tamavidin, and the like.

  The virus-like particle according to the present invention is obtained by using a kit or the like that obtains a virus-like particle containing a protein having self-organization ability using a known method and modifies it via a cysteine residue. A physiologically active molecule may be bound. Moreover, after bonding, a known means such as gel filtration may be employed as appropriate for the purification step.

After binding the bioactive molecule, a known cross-linking agent is used by a known method to bind the cysteine residue of the protein having self-organization ability contained in the virus-like particle to the bioactive molecule. A process of strengthening may be performed. Known crosslinking agents include BS _{3 and the} like.

  In addition to the above-mentioned bioactive molecule that binds to the cysteine residue, the virus-like particle according to the present invention further binds to a bioactive molecule (hereinafter sometimes referred to as a second bioactive molecule in the specification). Embodiments of virus-like particles having a protein with the ability to self-assemble are also included.

  As a virus-like particle of such an embodiment, an embodiment in which the lipid component constituting the lipid bilayer membrane of the virus-like particle and the second physiologically active molecule are bound, other than the cysteine residue of the protein having the self-organizing ability described above Examples include a mode in which the second physiologically active molecule binds via an amino acid residue or a sugar chain, and a mode in which the second physiologically active molecule is incorporated at a specific site of the protein having the self-organizing ability.

  The second bioactive molecule described above can be the same as the bioactive molecule modified via the cysteine residue described above.

  For example, as an embodiment in which an antibody binding domain is incorporated as the second physiologically active molecule in the N-terminal region of the above-mentioned protein having self-assembly ability, a protein having the self-assembly ability consisting of the amino acid sequence shown in SEQ ID NO: 2 is used. And virus-like particles. That is, such a protein having self-assembly ability has an antibody binding domain as a bioactive molecule via the N-terminal region of such protein and the thiol group of at least one cysteine residue of such protein. Also good.

  For example, if the second physiologically active molecule is bound to the sugar chain of the protein having the above-mentioned self-organization ability, it can be obtained by subjecting the terminal sugar residue of the sugar chain such as sialic acid to aldehyde formation. Can do.

The binding between the protein having self-organization ability and the second physiologically active molecule may be subjected to a treatment for strengthening the binding using a known crosslinking agent as described above. Known crosslinking agents include BS _{3 and the} like.

In addition, when the second physiologically active molecule is an antibody binding domain, an embodiment in which an antibody is bound to such a domain is also included in the virus-like particle according to the present invention. The virus-like particle according to the present invention also includes an embodiment in which a crosslinking treatment for strengthening the bond is performed as necessary. Known crosslinking agents include BS _{3 and the} like.

  A preferred embodiment of the virus-like particle according to the present invention includes a protein having self-assembly ability, (1) an antibody-binding domain in the N-terminal region of the protein, and the above-mentioned specific cysteine residue of the protein An embodiment in which an enzyme is bound via “(SH-HRP-labeled BNC-ZZ” in [Production Example 3] in the following Examples and “SH-ALP-labeled BNC-ZZ” in [Production Example 4]) (2) A mode in which an antibody-binding domain is bound to the N-terminal region of the protein and the antibody-binding domain is bound via the above-mentioned specific cysteine residue of the protein (in [Production Example 5] in the following Examples) “AGG-BNC-ZZ” corresponds to this.); (3) The above-mentioned identification of the protein having an antibody-binding domain in the N-terminal region of the protein An embodiment in which an antibody binding domain binds via a cysteine residue and an enzyme binds via the N-terminal or lysine residue of the protein ("HRP-labeled AGG-BNC-ZZ" in [Production Example 6] in the following Examples) "(Corresponding to this);) (4) An aspect in which the avidin compound is bonded via the above-mentioned specific cysteine residue of the protein (" BNC-SA "in [Production Example 8] in the Example below) (5) Aspects in which an avidin compound binds via the above-mentioned specific cysteine residue of the protein and an enzyme binds via a lysine residue of the protein (Production Examples in the Examples below) 8] corresponds to this.); (6) A mode in which the enzyme binds via the specific cysteine residue of the protein (described below) “HRP-labeled BNC-L” in [Production Example 9] of the example corresponds to this.); (7) The antibody-binding domain binds via the sugar chain of the protein, and An embodiment in which an enzyme binds via a cysteine residue (“SH-HRP-labeled BNC- (sugar chain) -AGG” in [Production Example 10] of the following Example corresponds to this)); (8) Examples include an embodiment in which an enzyme binds via the specific cysteine residue described above ([Production Example 11: HRP-labeled HVJ-E] in the following Examples corresponds to this).

  The immunological measurement method defined in the present specification is not particularly limited as long as it is a measurement method that employs an antigen-antibody binding action by an antibody as a measurement principle. For example, Western blotting method, ELISA method, immunochromatography method , Immunostaining method, EIA method, FIA method, and various modified methods based on these methods.

<Blocking agent>
The blocking agent according to the present invention is used in an immunological assay using the above-described virus-like particle according to the present invention. Such a blocking agent exhibits the effect of decreasing the background of data obtained in the above-described immunological measurement and / or sensitizing the detection signal, and can significantly increase the S / N ratio.

  Such an effect is not evaluated only by the obtained data, but is also evaluated by, for example, confirming the suppression of the adsorption of the above-mentioned virus-like particles to laboratory instruments widely used in immunological measurement methods.

  The virus-like particles and the immunological measurement method can be as described in detail in the above <virus-like particles>.

  The blocking agent according to the present invention contains hydroxyalkyl cellulose, polyvinyl alcohol, an ethylene oxide / propylene oxide copolymer, a copolymer composed of 2-methacryloyloxyethylphosphocholine, or two or more of these. .

  The hydroxyalkyl cellulose is not particularly limited, but preferably has an alkyl group having about 1 to 4 carbon atoms. For example, hydroxypropylcellulose, hydroxyethylcellulose, hydroxypropylmethylcellulose, hydroxyethylmethylcellulose, hydroxypropylethylcellulose, hydroxyethylethylcellulose and the like can be mentioned, among which hydroxypropylmethylcellulose is preferable.

  The hydroxyalkyl cellulose contained in the blocking agent according to the present invention may be a combination of two or more of the above-mentioned compounds. Specific acquisition methods include purchase from the market and production using known methods. Etc.

  The polyvinyl alcohol may be a polymer having vinyl alcohol as a monomer unit. Although the polymerization degree of polyvinyl alcohol is not particularly limited, it is usually preferably about 200 to 5,000, more preferably about 500 to 2,000.

  Further, since usually used polyvinyl alcohol is often obtained by saponifying polyvinyl acetate, it may have vinyl acetate as a monomer unit as described above. Based on such a viewpoint, the saponification degree (mol%) of the above-mentioned polyvinyl alcohol is usually about 80 to 98, preferably about 85 to 98.

  The polyvinyl alcohol contained in the blocking agent according to the present invention may be a combination of two or more of the above-mentioned compounds. Specific acquisition methods include purchase from the market, production using a known method, etc. Is mentioned.

  The ethylene oxide / propylene oxide copolymer is not particularly limited as long as it is a copolymer having ethylene oxide and propylene oxide as monomer units, and is preferably, for example, Pluronic (registered trademark) or an equivalent thereof. .

  More preferable ethylene oxide / propylene oxide copolymers include Pluronic L31, Pluronic L35, Pluronic L64, Pluronic P94, Pluronic F68, Pluronic F87, Pluronic F-127, Pluronic P-105 and the like (trademark of BASF). As an equivalent of these, poloxamer (trademark of ICI) can be mentioned. Among these, pluronic F-127 or pluronic P-105, its equivalent, etc. are the most preferable.

  The ethylene oxide / propylene oxide copolymer contained in the blocking agent according to the present invention may be a combination of two or more of the above-mentioned compounds. Specific methods for obtaining the market include those on the market (BSAF and ICI). Etc.) and production using a known method.

  The copolymer composed of 2-methacryloyloxyethylphosphocholine is not particularly limited as long as it is a copolymer having 2-methacryloyloxyethylphosphocholine and other monomer components as constituent units.

  More specifically, examples of the copolymer composed of the above-mentioned 2-methacryloyloxyethylphosphocholine include Lipidure (registered trademark), Biolipide (registered trademark), and equivalents thereof.

  Examples of preferred copolymers composed of 2-methacryloyloxyethylphosphocholine include, for example, lipid BL-405, lipid BL-203, lipid BL-1002, lipid BL-103, lipid BL-206, lipid BL-802, These equivalents and the like can be mentioned, and there is no particular limitation. Among them, lipid BL-206, lipid BL-802, equivalents thereof and the like are most preferable (in this text, for example, lipid BL-802 is lipid 802. And simplified description).

  The copolymer composed of 2-methacryloyloxyethylphosphocholine contained in the blocking agent according to the present invention may be a combination of two or more of the above-mentioned compounds. The purchase from (NOF Corporation), manufacture using a well-known method, etc. are mentioned.

  The content of the copolymer composed of hydroxyalkyl cellulose, polyvinyl alcohol, ethylene oxide / propylene oxide copolymer, or 2-methacryloyloxyethylphosphocholine contained in the blocking agent according to the present invention is not particularly limited. The total weight of the copolymer composed of hydroxyalkyl cellulose, polyvinyl alcohol, ethylene oxide / propylene oxide copolymer and 2-methacryloyloxyethylphosphocholine with respect to 100 parts by weight of the blocking agent is 0.001 to 100 parts by weight. It may be about a part. That is, a hydroxyalkyl cellulose, polyvinyl alcohol, an ethylene oxide / propylene oxide copolymer, or a copolymer itself composed of 2-methacryloyloxyethylphosphocholine may be used as the blocking agent according to the present invention.

  In the blocking agent according to the present invention, the hydroxyalkyl cellulose, polyvinyl alcohol, ethylene oxide / propylene oxide copolymer, 2-methacryloyloxy, which are used in the immunological measurement method as long as the above effects are not hindered. Components other than the copolymer composed of ethylphosphocholine may be included. Although it does not specifically limit with a specific other component, For example, antiseptic | preservative, a pH adjuster, a salt, surfactant, a stabilizer, etc. are mentioned. In addition, the blocking agent according to the present invention may be provided in a form included in water, a buffer solution, or the like that is widely used in advance in an immunological measurement method, and is provided as a solid form. You may provide in the aspect which can be dissolved in the above-mentioned water, a buffer solution, etc., and can be prepared and used at the time of use.

  Although the usage-amount of the blocking agent based on this invention is not specifically limited, For example, what is necessary is just about 0.0001-5 volume parts with respect to 100 volume parts of solutions, such as a buffer solution used in immunological measurement.

<Immunological measurement kit>
The immunological measurement kit according to the present invention includes the above-described virus-like particle according to the present invention and the above-described blocking agent according to the present invention.

  The virus-like particles and the blocking agent are as described in detail in the above <Blocking agent>. The immunological measurement can be the same as the immunological measurement method described in detail in the above <virus-like particle>.

  The immunological measurement kit according to the present invention may further contain a reaction vessel, a color developing / luminescent substrate, a reaction solution, a standard substance, a disposable instrument, a manual, and the like.

  In the immunoassay kit according to the present invention, the virus-like particle according to the present invention and the blocking agent according to the present invention are filled in separate containers (packs, bottles, etc.), respectively. It may be filled in the same container. In addition, the virus-like particles and the blocking agent may be provided in a form that is contained in water, a buffer solution, and the like. In particular, the blocking agent is provided in a powder form, and water, a buffer solution, and the like when performing immunological measurement. And may be provided in an embodiment that is appropriately prepared (diluted) at the time of use.

  Examples for explaining the present invention in more detail are shown below. However, it goes without saying that the present invention is not limited to the examples shown below.

[Production Example 1: Preparation of BNC-L]
BNC-L is a virus-like particle containing the HBsAg protein having the self-assembling ability consisting of the amino acid sequence shown in SEQ ID NO: 1, and was prepared by the method described in Japanese Patent No. 4085231 or Japanese Patent No. 4936272.

  Specifically, the yeast that expresses BNC-L prepared in Japanese Patent No. 4085231 is cultured, and the bacterial cell disruption solution obtained by disrupting the cultured bacterial cells using glass beads is cultured at 70 ° C. for 20 minutes. The sample was subjected to heat treatment. After the heat treatment, it was subjected to a centrifugation step, and the resulting supernatant was collected and then purified using a sulfate cellulofine column and a gel filtration column, and concentrated to a protein concentration of 0.2 mg / mL or more. L was obtained.

  In addition, it is reported that BNC-L containing a protein having the self-assembling ability consisting of the amino acid sequence shown in SEQ ID NO: 1 forms particles, and about 110 molecules of the protein per particle are included (Yamada et al. , Vaccine 19, 3154-3163, 2001).

[Production Example 2: Preparation of BNC-ZZ]
BNC-ZZ is a self-organization represented by SEQ ID NO: 2 having two antibody binding domains (Z domains) of protein A in tandem at the N-terminus of the HBsAg protein contained in BNC-L [Production Example 1]. A virus-like particle containing a protein having a chemical ability, which was prepared by the method described in Japanese Patent No. 4212921 and Japanese Patent No. 4936272.

  Specifically, the yeast that expresses BNC-ZZ prepared in Japanese Patent No. 4212921 is cultured, and the bacterial cell disruption solution obtained by disrupting the cultured bacterial cells using glass beads is maintained at 70 ° C. for 20 minutes. The sample was subjected to heat treatment. After the heat treatment, it was subjected to a centrifugation step, and the resulting supernatant was recovered, purified using a Porcine IgG column and a gel filtration column, concentrated to a protein concentration of 0.2 mg / mL or more, and BNC-ZZ. Got.

  In addition, it is estimated that the protein which has the self-organization ability which consists of an amino acid sequence shown to sequence number 2 is contained about 110 molecules per BNC-ZZ particle | grain from the analogy of said BNC-L. Further, when mixed with an antibody, BNC-ZZ forms a complex that retains the antigen-binding ability of the antibody by binding between the antibody-binding domain of the particle and the Fc domain of the antibody. The enzyme label of BNC-ZZ forms a similar complex. Hereinafter, the complex may be referred to as a mixed complex.

[Production Example 3: Preparation of HRP-labeled BNC-ZZ]
For BNC-ZZ [Production Example 2], a kit for labeling HRP via the SH group and a kit for labeling HRP via the NH ₂ group (Peroxidase Labeling Kit-SH and Peroxidase Labeling Kit-NH, respectively) ₂ ; Dojindo Chemical Co., Ltd.) and labeled with HRP according to these manuals, and then purified with a gel filtration column to obtain two types of HRP-labeled BNC-ZZ.

  In addition, it was confirmed that HRP labeling via SH groups contained about 110 HRP molecules per BNC-ZZ particle. That is, it is suggested that about one HRP molecule is crosslinked to the SH group of at least one cysteine residue in the protein consisting of the amino acid sequence shown in SEQ ID NO: 2 above.

In addition, BNC-ZZ used in the preparation of HRP-labeled BNC-ZZ was subjected to HRP labeling under mild conditions that did not affect the particle shape. Actually, as a result of measuring the particle diameter by the dynamic light scattering method, it was 58 nm after the labeling compared to 54 nm before the labeling, and a large difference was not recognized. Therefore, NH ₂ groups and SH groups HRP label, it is clear that the NH ₂ group and SH group derived from the amino acid residues exposed on both the particle surface.

If the SEQ ID NO: 2 shows the amino acid sequence of the protein contained in the BNC-ZZ based, if for example an NH ₂ group, NH ₂ group of the N-terminal or first, 43 th, 67 th, 70 th, 98 Via the NH ₂ group of the lysine residue side chain of any of the 1st, 112th, 113th, 121st, 125th, 128th, 156th, 170th, 171st, 179th, 308th, or 327th This suggests that it is HRP labeled.

  On the other hand, in the case of an SH group, for example, HRP labeling is performed via the SH group of any one of the cysteine residues at 293, 307, 310, 323, 324, 325, 333, or 335. It is suggested that.

Example 1
An experiment was conducted to measure the HRP activity of HRP-labeled BNC-ZZ prepared by the two methods shown in Production Example 3 above. SAT-blue solution (Dojindo Laboratories) was used as a substrate for HRP, absorbance at 492 nm (Abs 492 nm) was measured, and specific activity was determined using the amount of protein calculated by measuring absorbance at 280 nm.

As a result, the specific activity of BNC-ZZ HRP-labeled via the NH ₂ group (hereinafter sometimes referred to as NH ₂ -HRP-labeled BNC-ZZ) is 0.351 unit / μg (U / μg). On the other hand, the specific activity of HNC-labeled BNC-ZZ (hereinafter sometimes referred to as SH-HRP-labeled BNC-ZZ) via the SH group was 0.844 U / μg. That is, NH ₂ -HRP labeled BNC-ZZ showed only about 41% of the activity of SH-HRP labeled BNC-ZZ. In addition, unit shows the specific increase value of Abs 492nm value when it measures using SAT-blue which is a substrate on said conditions.

This result suggests that there are fewer NH ₂ groups accessible at a level at which a protein molecule called HRP can be labeled compared to SH groups. In addition, since more HRP molecules can be labeled through the SH group, it is clear that this labeling method has the potential to use HRP-labeled BNC-ZZ as a more sensitive antibody detection probe. It became.

[Production Example 4: Preparation of ALP-labeled BNC-ZZ]
BNC-ZZ [Production Example 2] kit for ALP labeling via SH group and kit for ALP labeling via NH ₂ group (Alkaline Phosphatase Labeling Kit-SH and Alkaline Phosphatase Labeling Kit- NH ₂ ; both of which are Dojindo Chemical Co., Ltd.) and ALP-labeled according to these manuals, and then purified by gel filtration column to obtain two types of ALP-labeled BNC-ZZ.

  Similarly to the HRP-labeled BNC-ZZ prepared in Production Example 3, the BNC-ZZ used in the preparation of the ALP-labeled BNC-ZZ was subjected to ALP labeling under conditions such that the particle shape was not destroyed. .

Therefore, it is suggested that each ALP-labeled BNC-ZZ is also ALP-labeled on the NH ₂ group or SH group in the same amino acid residue as the above-mentioned HRP-labeled BNC-ZZ.

Example 2
Experiments were performed to measure the ALP activity of ALP-labeled BNC-ZZ prepared by the two methods shown in Production Example 4 above. Using pNPP (Sigma Fast p-nitrophenyl phosphate tablets) as a substrate for ALP, the absorbance at 405 nm (Abs 405 nm) was measured, and the specific activity was determined using the amount of protein calculated by measuring the absorbance at 280 nm. .

As a result, the specific activity of the ALP enzyme of BNC-ZZ ALP-labeled through the NH ₂ group (hereinafter sometimes referred to as NH ₂ -ALP-labeled BNC-ZZ) is 3.56 U / μg, Thus, ALP-labeled BNC-ZZ (hereinafter sometimes referred to as SH-ALP-labeled BNC-ZZ) via the SH group was 5.61 U / μg. That is, NH ₂ -ALP labeled BNC-ZZ showed only about 60% of the activity of SH-ALP labeled BNC-ZZ. The unit indicates a specific increase in Abs 405 nm value when the substrate pNPP is reacted under the above conditions.

  As a result, it can be seen that more ALP molecules can be labeled via the SH group, and this labeling method has the potential to use ALP-labeled BNC-ZZ as a more sensitive antibody detection probe. It became clear.

[Production Example 5: Preparation of AGG-BNC-ZZ]
A protein comprising the amino acid sequence shown in SEQ ID NO: 6 containing one binding domain derived from protein A having the Z domain and two binding domains derived from protein G was prepared in E. coli (hereinafter, this may be referred to as AGG). ). EMCS (Dojindo) was added to the AGG protein solution to introduce a maleimide group into the amino group of AGG.

  BNC-ZZ [Production Example 2] is subjected to a reduction treatment using TCEP (Thermo Scientific), and the crosslinking treatment is performed by incubating the reduced BNC-ZZ and AGG into which a maleimide group has been introduced. BNC-ZZ was obtained. That is, AGG-BNC-ZZ is a virus-like particle containing a protein having the amino acid sequence represented by SEQ ID NO: 2 and having AGG bound thereto via a cysteine residue in the protein.

[Production Example 6: Preparation of HRP-labeled AGG-BNC-ZZ]
HRP was labeled on AGG-BNC-ZZ [Production Example 5] using Peroxidase labeling Kit-NH ₂ according to the manual to obtain HRP-labeled AGG-BNC-ZZ.

Example 3
An experiment was conducted to compare the HRP enzyme activity of HRP-labeled AGG-BNC-ZZ [Production Example 6] with the enzyme activity of SH-HRP-labeled BNC-ZZ [Production Example 3]. A fixed amount of both was taken, a TMB solution (One-Step TMB Ultra, Thermo Scientific) as a substrate of HRP was added thereto, and Abs 450 nm was measured.

As a result, it was found that the HRP specific activity of HRP-labeled AGG-BNC-ZZ was about 1/3 that of HRP-labeled BNC-ZZ. Judging from the results of Example 2 in which BNC-ZZ was labeled with HRP via NH ₂ group or SH and HRP activity was examined, AGG-BNC-ZZ was mediated through NH ₂ group with almost the same efficiency as BNC-ZZ. It became clear that HRP labeling was possible.

[Production Example 7: Preparation of HRP-labeled BNC-ZZ / rabbit-derived anti-mouse IgG antibody complex]
As described in Production Example 2, when BNC-ZZ and the antibody are mixed, a mixed complex is formed. In this mixed complex, since the binding between the Fc region of the antibody and the antibody binding site of BNC-ZZ is reversible, the exchange of the bound antibody occurs in the presence of a plurality of antibodies. Therefore, if this binding is made irreversible, it is considered that the binding ability of the bound antibody can be imparted to BNC-ZZ. Thus, a cross-linked antibody binding domain and antibody was produced as follows. That is, SH-HRP-labeled BNC-ZZ [Production Example 3] and a rabbit-derived anti-mouse IgG antibody (Bethyl) are mixed to form a complex of both, and BS ₃ (Dojin Chemical), which is a cross-linking agent, is terminated. The concentration was 0, 50, 200, 400, or 1000 μM. Subsequently, excess rabbit-derived anti-mouse IgG antibody was removed using Protein A Sepharose resin (GE Healthcare) to obtain an HRP-labeled BNC-ZZ / rabbit-derived anti-mouse IgG antibody complex.

Example 4
An experiment was conducted to measure the HRP activity of the HRP-labeled BNC-ZZ / rabbit-derived anti-mouse IgG antibody complex [Production Example 7]. HRP activity was measured by the method described in Example 3 using an HRP-labeled BNC-ZZ / antibody complex. The results are shown in Table 1. Abs 450 nm shows almost the same value at any crosslinker concentration, indicating that the activity of HRP hardly changes.

Example 5
Competition experiments were performed to evaluate the binding mode of the antibody bound to the SH-HRP labeled BNC-ZZ / antibody complex. IgG having no antigenic properties (hereinafter sometimes referred to as control mouse-derived IgG) obtained by purifying from normal mouse serum with protein A / G sepharose (GE Healthcare) at various concentrations in each well of the ELISA plate. Added and solidified. Then, it blocked using k-Block-e (made by a vehicle company).

  HRP-labeled BNC-ZZ / rabbit-derived anti-mouse IgG antibody complex [Production Example 7] and control rabbit-derived IgG (prepared in the same manner as above, manufactured in-house) were mixed and incubated, and then the mixture was immobilized. After adding and reacting with each well, Abs 450 nm was measured in the same manner as in Example 3. The result is shown in FIG.

HRP-labeled BNC-ZZ and anti-mouse IgG rabbit-derived antibody complex having a cross-linking agent concentration of 0 M, that is, when no cross-linking treatment is performed, in the presence of control rabbit-derived IgG, compared to non-coexistence The response to immobilized mouse-derived IgG was reduced to 70% in all concentration ranges. When the HRP-labeled BNC-ZZ / rabbit-derived anti-mouse IgG antibody complex was subjected to a cross-linking treatment using 50 μM BS ₃ , in the presence of control rabbit-derived IgG, a slight amount of the immobilized mouse-derived IgG was obtained. A decrease in reaction is seen, indicating that reversible binding remains. In addition, in the complex subjected to the crosslinking treatment using BS ₃ at a concentration of 200 μM, there was no change in the response to mouse-derived IgG even when control rabbit-derived IgG was added. That is, the HRP-labeled BNC-ZZ / rabbit-derived anti-mouse IgG antibody complex cross-linked with 200 μM BS ₃ does not cause a change in the antibody binding thereto, and indicates that a complex is formed by irreversible binding. .

From the above, it was revealed that the HRP-labeled BNC-ZZ / rabbit-derived anti-mouse IgG antibody complex crosslinked with BS ₃ at a concentration of 200 μM or more can form a complex due to irreversible binding.

Example 6
An experiment was conducted to evaluate the residual antibody binding activity and reversible antibody binding activity of the HRP-labeled BNC-ZZ / rabbit-derived anti-mouse IgG antibody complex [Production Example 7]. In the method for producing an HRP-labeled BNC-ZZ / rabbit-derived anti-mouse IgG antibody complex treated with various concentrations of the cross-linking agent shown in Production Example 7, instead of the rabbit-derived anti-mouse IgG antibody, a control rabbit-derived IgG and A composite was prepared. The concentrations of BS ₃ used are 0 μM, 50 μM, and 200 μM. These complexes were added to each well of the ELISA plate and solidified, and further blocked using k-Block-e (manufactured by Vehicle). Next, an ALP-labeled rabbit-derived anti-mouse IgG antibody (Invitrogen) diluted to 1/10000, 1/5000, and 1/1000 was added to each well for reaction, and after washing, Abs 405 nm was obtained in the same manner as in Example 2. It was measured. The results are shown in FIG.

In BNC-ZZ and HRP-labeled BNC-ZZ, the ALP-labeled rabbit antibody increased the ALP enzyme activity depending on the amount added, and when the 1/1000 dilution was added, the ALP enzyme activity was more than 4 times that of the non-added one. This indicates that the ALP-labeled rabbit antibody is bound to the antibody binding site of BNC-ZZ or HRP-labeled BNC-ZZ. On the other hand, a mixed complex formed by premixing HRP-labeled BNC-ZZ and control rabbit-derived IgG (ZZ-HRP 0 in the figure) or a complex that was crosslinked by adding 50 μM of BS ₃ (in the figure ZZ-HRP 50) showed an ALP activity of 170% in the former and 137% in the latter when 1/1000 dilution was added compared to the case without addition. These conjugates indicate that ALP-labeled rabbit antibodies can bind, while in the case of conjugates of BS ₃ crosslinked with 200 μM (ZZ-HRP 200 in the figure), ALP-labeled rabbit antibodies The binding of was not seen.

From the above, in the case of a complex of HRP-labeled BNC-ZZ and antibody, there is no antibody binding activity remaining in HRP-labeled BNC-ZZ and no reversible binding when crosslinked at a concentration of BS ₃ of 200 μM or higher. It is shown that.

[Production Example 8: Preparation of HRP-labeled BNC-SA / anti-mouse IgG antibody complex]
A maleimide group (MAL group) was introduced into the amino group of streptavidin (SA; manufactured by Thermo Scientific) using EMCS. Subsequently, BNC-L [Production Example 1] is incubated with SA into which the MAL group has been introduced to carry out a crosslinking reaction to obtain SA-labeled BNC-L (hereinafter sometimes referred to as BNC-SA). It was. That is, BNC-SA is a virus-like particle containing a protein having the amino acid sequence shown in SEQ ID NO: 1 and having SA bound thereto via a cysteine residue of the protein. Further, HRP was crosslinked to the obtained BNC-SA using Peroxidase Labeling Kit-NH ₂ to obtain HRP-labeled BNC-SA. In addition, a biotinylated anti-mouse IgG antibody derived from rabbit was obtained using Biotin Labeling Kit-NH ₂ (Dojin Chemical). The HRP-labeled BNC-SA and rabbit-derived biotinylated anti-mouse IgG antibody were mixed in an equal amount as a protein amount to prepare an HRP-labeled BNC-SA / rabbit-derived anti-mouse IgG antibody complex.

Example 7
An experiment was conducted to measure the HRP activity of the HRP-labeled BNC-SA / anti-mouse IgG antibody complex. The HRP activity of the HRP-labeled BNC-SA / rabbit-derived anti-mouse IgG antibody complex [Production Example 8] was measured in the same manner as in Example 1.

As a result, it was found that the HRP activity of the HRP-labeled BNC-SA / rabbit derived anti-mouse IgG antibody complex was about 1/8 that of the control SH-HRP-labeled BNC-ZZ. From the result of Example 1, since the HRP activity of NH ₂ -HRP-labeled BNC-ZZ is about 1 / 2.4 of SH, the HRP activity of HRP-labeled BNC-SA / rabbit-derived anti-mouse IgG antibody is NH ₂ compared to -HRP labeled BNC-ZZ If it can be determined that about one third.

Example 8
Experiments were conducted to examine the adsorption of BNC-ZZ to the container and the effect of the blocking agent. A solution prepared by dissolving BNC-ZZ [Production Example 2] in PBS to 300 ng / mL was dispensed into a polyethylene microtube, and various blocking agents shown in Table 2 were added thereto. Each tube was sealed and left at room temperature for 4 days while rotating the container, and then the amount of BNC-ZZ remaining in the solution was measured. For the measurement, an ELISA kit (HB Pre-S1 Antigen Quantitative ELISA Kit, Rapid, manufactured by Vehicle) for measuring the Pre-S1 region on the particle surface of BNC-ZZ was used and measured according to the attached manual. The results are shown in Table 2.

  In the case of PBS alone, 1.3% BNC-ZZ remained in the solution, and it was found that most was adsorbed to the container. When various blocking agents were added thereto, all showed inhibitory effects, but the most effective were skim milk, Block Ace (Dainippon Pharmaceutical), Pluronic F-127, and Lipidure 802.

Example 9
Experiments were conducted to examine the adsorption of HRP-labeled BNC-ZZ to the container and the effect of the blocking agent. A PBS solution of SH-HRP-labeled BNC-ZZ [Production Example 3] having a final concentration of 300 ng / mL containing various blocking agents shown in Table 3 was prepared, and each was a polyethylene tube without surface treatment, MPC (2-methacryloyloxyethyl phosphorylcholine). Placed in treated tubes and glass tubes. All tubes were sealed and left at 4 ° C. for 2 days while rotating, and the HRP activity contained in the solution was measured by the following method. That is, 2 μg / mL porcine IgG was added to each well of a 96-well microplate for ELISA to immobilize. Next, 1% Block Ace was added and blocked. Here, the HRP-labeled BNC-ZZ solution left in the above-mentioned various tubes was added to each well and allowed to bind to the immobilized porcine IgG. Next, a TMB solution (1-Step TMB slow: Thermo Scientific) was added to each well, and then the Abs 450 nm value was measured with a plate reader. The residual rate of HRP-labeled BNC-ZZ in each sample was calculated based on the Abs 450 nm value of what was obtained by diluting the sample that had been similarly stored in the MPC-treated tube at 500 μg / mL to 300 ng / mL as a control. Table 3 shows the results.

  When a solution containing no blocking agent (PBS in the table) was placed in an untreated plastic tube, 78% of the adsorption occurred in 2 days and 48% of the glass tube. Adsorption was suppressed in the MPC-treated tube, but 10% adsorption remained. When various blocking components were added here, an inhibitory effect was observed, but BSA, HPMC, Pluronic F-127 and Pluronic P-105 showed good results.

Example 10
Experiments were conducted to examine the effect of BNC-ZZ adsorption and blocking agent on the PVDF membrane. The PVDF membrane is cut into small pieces, and is subjected to blocking treatment by immersing in PBS containing various concentrations of blocking agents shown in Table 4 at room temperature for 1 hour, and washed with PBS-T three times to bind to the membrane. No blocking agent was removed. Next, a reaction solution prepared by dissolving BNC-ZZ [Production Example 2] at a concentration of 300 ng / mL in PBS-T containing various concentrations of the blocking agent shown in Table 4 and the above-mentioned blocked PVDF membrane at room temperature for 1 hour. After that, the mixture was reacted with an HRP-labeled rabbit antibody dissolved in PBS-T at room temperature for 20 minutes.

  After completion of the reaction, it is washed 5 times with PBS-T, reacted with ECL Prime (GE Healthcare), which is an HRP luminescence substrate, and luminescence is emitted for 20 minutes using a luminescence detection device (ChemiDoc XRS, Bio-Rad). It measured by exposing.

  In addition, what performed the same process with PBS-T which does not contain a blocking component as control was utilized. The results are shown in Table 4.

  It was clear that the PVDF membrane treated only with PBS containing no blocking agent was entirely black, and a large amount of BNC-ZZ was adsorbed on the PVDF membrane.

  On the other hand, when blocking treatment is applied to the PVDF membrane using various blocking agents and the same blocking agent is contained in the BNC-ZZ solution to be further reacted, adsorption of BNC-ZZ to the PVDF membrane with any blocking agent Was hardly recognized.

  From the above, it was clarified that all blocking agents used in this experiment exhibited a blocking effect when used in both the blocking treatment of the PVDF membrane and the blocking treatment performed by adding it to the reaction solution. .

Example 11
An experiment was conducted to examine the effect of the blocking agent and the adsorption of HRP-labeled BNC-ZZ to the PVDF membrane. A solution (membrane blocking solution) prepared by dissolving 5% skim milk, 10% skim milk + 3% fish gelatin (funakoshi), 4% block ace and 5% bovine serum albumin in TBS was prepared. The PVDF membrane cut into small pieces was subjected to a blocking treatment using the membrane blocking solution. The membrane blocking treatment was controlled as an untreated PVDF membrane.

  Next, TBS-T containing SH-HRP-labeled BNC-ZZ [Production Example 3] is prepared, and 1% skim milk, 1% block ace, 1% BSA, or 2% gelatin is added, A reaction blocking solution was prepared, and the reaction blocking solution was added to the PVDF membrane. As a control, TBS-T containing SH-HRP-labeled BNC-ZZ was used. After the addition, a PVDF membrane that was appropriately washed was detected in the same manner as in Example 10. The results are shown in Table 5.

  When the PVDF membrane was not subjected to blocking treatment and TBS-T containing HRP-labeled BNC-ZZ was added, the entire image became black and the signal intensity score was +++++. This indicates that a large amount of HRP-labeled BNC-ZZ was adsorbed on the PVDF membrane. On the other hand, when TBS-T containing HRP-labeled BNC-ZZ was added to PVDF membranes treated with various membrane blocking solutions, the adsorption of HRP-labeled BNC-ZZ to the PVDF membrane was suppressed, but still an obvious adsorption reaction It was observed. In addition, when the PVDF membrane is treated with a membrane blocking solution and each blocking solution is added, the reaction blocking solution containing BSA is excluded and the adsorption of HRP-labeled BNC-ZZ to the PVDF membrane is strongly suppressed compared to the control. It was done. In the reaction blocking solution containing BSA, unlike the other reaction blocking solutions, the degree of signal intensity obtained from the PVDF membrane was high regardless of the type of blocking treatment of the PVDF membrane.

  The above results show that the blocking treatment to the PVDF membrane is important for suppressing the adsorption of HRP-labeled BNC-ZZ to the PVDF membrane, and the type of the reaction blocking solution is more important than that. All blocking solutions to the PVDF membrane had the same effect, but as the reaction blocking solution, block ace, skim milk, and fish gelatin had almost the same effect, but BSA was found to be weak.

Example 12
Experiments were conducted to examine the effects of HRP-labeled BNC-ZZ adsorption on the PVDF membrane and chemical blocking agents. Various blocking agents shown in Table 6 were dissolved so as to have a concentration of 1% in TBS. As a control, TBS in which 5% skim milk was dissolved in TBS was prepared. These were used to block the PVDF membrane, and then reacted with SH-HRP-labeled BNC-ZZ [Production Example 3], and then evaluated in the same manner as in Example 11. The results are shown in Table 6.

  In the PVDF membrane subjected to the blocking treatment with 5% skim milk, light blackening was observed in the entire membrane, and the adsorption of HRP-labeled BNC-ZZ to the membrane was not completely suppressed. Among the compounds studied, Pluronic F-127, Pluronic P-105, HPMC, PVA2000, PVA500, Lipidure 206, and Lipidure 802 showed almost no film blackening and a strong suppression effect was observed.

Example 13
Experiments were performed to effect the effect of HRP-labeled BNC-ZZ in Western blot. From the results of Examples 11 and 12, when SH-HRP-labeled BNC-ZZ is used in a PVDF membrane, Pluronic F-127, Pluronic P-105, HPMC, PVA2000, PVA500, Lipidure206 and Lipidure802 are used as blocking agents. Then, adsorption | suction to the film | membrane of HRP label | marker BNC-ZZ was suppressed, and when the blocking agent was further contained in the reaction liquid of HRP label | marker BNC-ZZ from the result of Example 10, it was assumed that an effect is still higher.

  Therefore, the effect when these blocking agents were contained in the reaction solution was examined by Western blotting of a HuH7 cell extract using an anti-vimentin antibody and SH-HRP-labeled BNC-ZZ [Production Example 3]. The detection method was the same as in Example 10. The membrane was blocked with 5% skimmed milk, and various blocking agents shown in Table 7 below having a final concentration of 1% were used for blocking during the reaction.

  A control experiment with 1% skim milk added resulted in a light blackening of the entire membrane as background. On the other hand, when Pluronic F-127, Pluronic P-105, HPMC, PVA2000, PVA500, Lipidure 206 and Lipidure 802 are added to the reaction solution, there are some differences in the effect depending on the type of blocking agent. The ground was lower than the control and was found to be effective in suppressing adsorption. On the other hand, the signal intensity of the antibody-specific band hardly changed with any blocking agent.

  From the above results, it is possible to suppress nonspecific adsorption of HRP-labeled BNC-ZZ by adding a blocking agent to the reaction solution of HRP-labeled BNC-ZZ, and at that time, antibody-specific signals are retained. I understood.

Example 14
Experiments were conducted to examine the effect of various blocking agents on the specific binding of probes in ELISA. Control mouse-derived IgG (polyclonal) was immobilized on an ELISA plate. As a control, an ELISA plate on which control mouse-derived IgG was not immobilized was prepared, and a plate subjected to blocking treatment with k-Block-e was prepared.

  Various probes (BNC-ZZ [Production Example 2], SH-HRP-labeled BNC-ZZ [Production Example 3], HRP-labeled AGG-BNC-ZZ [Production Example 6], HRP-labeled BNC-ZZ / rabbit) Anti-mouse IgG antibody complex [Production Example 7] and HRP-labeled BNC-SA / rabbit-derived anti-mouse IgG antibody complex [Production Example 8]) were added at a constant concentration and solidified with control mouse-derived IgG. The binding reaction was observed.

  In addition, various blocking agents shown in Table 8 were used together with various probes so that the final concentration was 0.1%. As a blocking agent control, PBS-T containing casein so as to have a final concentration of 0.2% was used.

  For BNC-ZZ, an HRP-labeled rabbit-derived control IgG antibody was simultaneously added, washed after the reaction, and Abs 450 nm was measured in the same manner as in Example 9. For each probe, the specific reaction is obtained by subtracting 100% of the reaction in the reaction solution with immobilized antibody and no blocking component as the blank value for each probe. It was. The results are shown in Table 8.

  When the reaction between the antibody and the probe was performed without adding any blocking component, the non-specific binding was very high and almost no specific reaction was observed. In casein used as a control, SH-HRP-BNC-ZZ showed a considerable specific reaction, but the response to other probes was low. Among blocking agents, HPMC and PVA showed low specific reaction, but others showed good specific reaction, although the size was different depending on the probe.

Example 15
Next, experiments were conducted to examine the effect of various probes on adsorption to the ELISA plate and the specific reaction of various blocking agents by changing the concentration of the blocking agent used in Example 14. The GFP protein prepared using E. coli was immobilized on an ELISA plate and blocked using k-Block-e.

  Various probes (SH-HRP-labeled BNC-ZZ [Production Example 3] and rabbit-derived anti-GFP antibody mixed complex added with various blocking agents on this plate; HRP-labeled AGG-BNC-ZZ [Production Example 6] and rabbit) Mixed anti-GFP antibody complex; HRP-labeled BNC-ZZ / rabbit-derived anti-GFP antibody complex) was added, and the binding reaction with the antigen was observed. Various blocking agents shown in Table 9 were examined under three conditions such that the final concentrations were 0.01%, 0.05%, and 0.1%, respectively.

The HRP-labeled BNC-ZZ / rabbit-derived anti-GFP antibody complex was prepared using a rabbit-derived anti-GFP antibody instead of the rabbit-derived anti-mouse IgG antibody with the concentration of BS ₃ in Production Example 7 being 1000 μM. As a control, the above-described control rabbit-derived IgG was used in place of the anti-GFP rabbit antibody, and Abs 450 nm was measured by the same method as shown in Example 9.

  The obtained results were obtained by dividing each signal value by each noise value with the value obtained in the absence of anti-GFP antibody as the noise value and the value obtained in the presence of anti-GFP antibody as the specific signal value. The numerical values are shown in Table 9 as the S / N ratio.

  Various blocking agents show different S / N ratios for each probe, and high blocking agent concentration shows a high S / N ratio. Conversely, a low blocking agent concentration shows a high S / N ratio. Some also showed. It was suggested that these blocking agents are effective when using probes such as BNC-ZZ in ELISA if they are used at appropriate concentrations.

Example 16
Experiments were conducted to evaluate the antibody binding activity of HRP-labeled BNC-ZZ. Antibody binding activities of NH ₂ -HRP-labeled BNC-ZZ and SH-HRP-labeled BNC-ZZ prepared in Production Example 3 were compared by ELISA. Control rabbit-derived IgG was immobilized on an ELISA plate, and each well was blocked with 1% Block Ace. HRP-labeled BNC-ZZ solutions having various concentrations of 0, 9.375, 18.75, 37.5, 75, 150, 300, 600 ng / mL as BNC-ZZ protein amounts were prepared, and the final concentration was 0.05. % PBS-T added so as to contain Pluronic F-127 was added to the well and allowed to react. After washing,
Abs 450 nm was measured by the method shown in Example 9. The result is shown in FIG.

At all concentrations, towards measurements obtained in SH-HRP-labeled BNC-ZZ was higher than that of _NH 2-HRP labeled BNC-ZZ. In particular, at concentrations of 300 ng / mL and 600 ng / mL, the measured values obtained with SH-HRP-labeled BNC-ZZ are approximately 2.1 to 2.7 times higher than those of NH ₂ -HRP-labeled BNC-ZZ. It was. This magnification is almost the same level as the difference in HRP specific activity shown in Example 1 above (the former is 0.844 and the latter is 0.351 U / μg, which is about 2.4 times higher). It was suggested that the HRP-labeled BNC-ZZ was bound to almost the maximum amount of the immobilized antibody at these concentrations. On the other hand, at a concentration lower than this, the magnification of the measured values obtained with SH-HRP-labeled BNC-ZZ and NH ₂ -HRP-labeled BNC-ZZ is high, and on average when the HRP-labeled BNC-ZZ concentration is 37.5 ng or less. It was about 7.24 times higher. This indicates that SH-HRP-labeled BNC-ZZ bound more to the immobilized antibody in the low concentration range, and considering that the specific activity of HRP is about 2.4 times different, SH -HRP labeled BNC-ZZ than _NH 2 -HRP labeled BNC-ZZ, binding affinity for antibodies suggests that strong about 3 times. Labeling via the NH ₂ group is performed mainly targeting lysine residues of the BNC-ZZ protein that constitutes the particle, but since there are many lysine residues at the antibody binding site, labeling via the NH ₂ group Is thought to affect antibody binding sites and inhibit antibody binding. On the other hand, since the SH group is present at an externally exposed site in the transmembrane region of the BNC-ZZ protein, it can be considered that the antibody binding ability is not affected when the SH group is targeted.

  Therefore, when labeling BNC-ZZ with HRP, labeling via the SH group can make more use of the antibody binding ability of BNC-ZZ, and also has higher HRP activity. It can be concluded that labeling via the SH group is far superior, since it can form a label approximately 7.2 times higher. Hereinafter, unless otherwise specified, the HRP-labeled BNC-ZZ is via an SH group.

Example 17
Experiments were conducted to examine the application of HRP-labeled BNC-ZZ in ELISA. An ELISA plate immobilized with Pre-S2 (product number BCL-AGS2-21), a peptide of the surface antigen of hepatitis B virus, was blocked with k-Block-e, and various concentrations of anti-Pre -S2 mouse antibody (Special Immunology Laboratory, 2APS42) was added. Subsequently, HRP-labeled anti-mouse antibody, HRP-labeled BNC-ZZ [Production Example 3] dissolved in PBS-T to which Pluronic F-127 was added so that the final concentration was 0.05%, or these at the same concentration The combined materials were added and reacted, and after washing, Abs 450 nm was measured in the same manner as in Example 9. The results are shown in FIG.

  In the figure, when using HRP-labeled BNC-ZZ as shown by the white triangle mark “HRP-ZZ”, compared to the case of detecting with the anti-mouse antibody as shown by the black circle mark “2nd IgG” in the figure. The sensitivity has increased by about 10 times, and it has been clarified that when both of these are used in combination, the sensitivity increases by about 30 times as indicated by the black square “HRP-ZZ + IgG”.

Example 18
In the same manner as in Example 17, antibody detection type ELISA of HRP-labeled BNC-ZZ was examined. The recombinant protein of Leishmania parasite and the control human antiserum used in this experiment were both provided by Aichi Medical University, Department of Infection and Immunology. An ELISA plate on which a recombinant protein of a protozoan that is a Leishmania pathogen was immobilized instead of Pre-S2 was blocked with k-Block-e, and various concentrations of control human antiserum were added. Next, HRP-labeled anti-human IgG antibody, HRP-labeled BNC-ZZ [Production Example 3] dissolved in PBS-T to which Pluronic F-127 has been added so that the final concentration is 0.05%, or these at the same concentration Those used in combination were added and reacted, and after washing, Abs 405 nm was measured with ABTS (1-STEP ABTS, Thermo Scientific) as a substrate. The results are shown in FIG. In addition, the measured value represented the antibody titer as Unit / mL.

  In the figure, when HRP-labeled BNC-ZZ is used as shown by the white square mark “HRP-ZZ”, when detected with an anti-human IgG antibody as shown by the black triangle mark “2nd IgG” in the figure The sensitivity was increased by a factor of 10 compared to the figure, and as shown by the black circle “HRP-ZZ + IgG” in the figure, it was revealed that when both of these were used in combination, the sensitivity increased by about 30 times.

  From the results shown in Examples 17 and 18 above, it was found that HRP-labeled BNC-ZZ is useful for high-sensitivity detection in an antibody detection type ELISA, and can be detected with higher sensitivity especially when it is coexisted with a detection antibody. It was.

Example 19
The practical measurement in ELISA of HRP-labeled BNC-ZZ was examined. The ELISA plate immobilized with GFP protein was blocked with k-Block-e. An anti-GFP mouse IgG antibody with a known concentration was used as a standard, and a mouse anti-GFP antiserum diluted 100 times or more as a sample was added to each well of the ELISA plate. Next, HRP-labeled BNC-ZZ dissolved in PBS with Pluronic F-127 added to a final concentration of 0.05% [Production Example 3], a mixture of rabbit-derived anti-mouse IgG antibody and HRP-labeled BNC-ZZ The complex or rabbit-derived HRP-labeled anti-mouse IgG antibody was added and reacted, and after washing, Abs 450 nm was measured in the same manner as in the method shown in Example 9. The measured values were calculated based on the respective calibration curves obtained with the standard antibody, with the concentration of anti-GFP mouse IgG in the antiserum being mean ± std ( ng / mL, n = 3).

As a result, the values measured with HRP-labeled BNC-ZZ, the mixed complex of HRP-labeled BNC-ZZ and anti-mouse IgG were 15613 ± 936 and 15403 ± 1192 , respectively, and quantified with HRP-labeled anti-mouse IgG antibody 15400 ± It was clear that it was in good agreement with 109 and had sufficient quantification.

Example 20
Experiments were conducted to evaluate the antibody binding activity of ALP-labeled BNC-ZZ. An ELISA plate solid-phased using control rabbit-derived IgG was blocked with 1% Block Ace, and NH ₂ -ALP-labeled BNC-ZZ or SH-ALP-labeled BNC-ZZ prepared in Preparation Example 4 was added to each well. Was converted into the amount of BNC-ZZ protein, and 50 μL each of 0, 9.375, 18.75, 37.5, 75, 150, 300, 600 ng / mL was added to each well, washed, and washed in the same manner as in Example 2. Abs 405 nm was measured. Pluronic F-127 with a final concentration of 0.05% was used together with ALP-labeled BNC-ZZ. The results are shown in FIG.

Better measurements concentration obtained in SH-ALP-labeled BNC-ZZ of all BNC-ZZ was higher than that of _NH 2 -ALP labeled BNC-ZZ. At a concentration of 600 ng / mL, the measured value obtained with SH-ALP labeled BNC-ZZ was about 1.7 times higher than that of NH ₂ -ALP labeled BNC-ZZ. This magnification is almost the same level as the difference in ALP specific activity between the two (the former is 5.61 and the latter is 3.56 units / μg, which is 1.6 times higher), and any ALP-labeled BNC-ZZ It was suggested that almost the maximum amount was bound to the immobilized antibody. On the other hand, at concentrations lower than this, the magnification of the measured values obtained with SH-ALP-labeled BNC-ZZ and NH ₂ -ALP-labeled BNC-ZZ is high, and an average of 3.7.5 ng at a BNC-ZZ concentration of 37.5 ng or less. It was about 6 times higher.

This suggests that more SH-ALP-labeled BNC-ZZ bound to the immobilized antibody in the low concentration range. Considering that the specific activity of ALP differs by about 1.6 times, it is estimated that SH-ALP-labeled BNC-ZZ has a 2.3-fold stronger binding affinity for the antibody than NH ₂ -ALP-labeled BNC-ZZ. It is considered that the reason why the antibody binding ability is decreased in the labeling via the NH ₂ group than in the case of via the SH group is the same cause as in the case of the HRP labeling described in Example 16 above.

Example 21
Experiments were conducted to examine the application of ALP-labeled BNC-ZZ to ELISA. An ELISA plate immobilized with IgG from a control mouse (polyclonal) was blocked with 0.5% casein. Various concentrations of SH-ALP-labeled BNC-ZZ [Production Example 4] or ALP-labeled rabbit-derived anti-mouse IgG antibody were added to each well for reaction. After washing, Abs 405 nm was measured in the same manner as in Example 2.

  Pluronic F-127 was used together with ALP-labeled BNC-ZZ so that the final concentration was 0.05%. The result is shown in FIG.

  When ALP-labeled BNC-ZZ was used, a reaction much higher than that of an ALP-labeled antibody was observed, suggesting that ALP-labeled BNC-ZZ is useful for highly sensitive antibody detection.

Example 22
Experiments were conducted to evaluate the antibody binding activity of HRP-labeled AGG-BNC-ZZ. An ELISA plate was immobilized using control pig-derived IgG (manufactured in-house prepared by the method of Example 5), and then blocked with 0.5% casein. The various concentrations of HRP-labeled AGG-BNC-ZZ [Production Example 6] or NH ₂ -HRP-labeled BNC-ZZ [Production Example 3] shown in the horizontal axis of FIG. Thereafter, Abs 450 nm was measured in the same manner as in Example 9. Pluronic F-127 was added to the probe so that the final concentration was 0.05%. The results are shown in FIG.

  As shown in Example 3, the HRP activity of HRP-labeled AGG-BNC-ZZ is known to be 1/3 that of HRP-labeled BNC-ZZ. In addition, HRP-labeled AGG-BNC-ZZ should show a 1/3 reaction of HRP-labeled BNC-ZZ with respect to solid-phased porcine-derived IgG.

  However, HRP-labeled AGG-BNC-ZZ actually had a reaction of about 1 / 1.1-1.2.4 compared with HRP-labeled BNC-ZZ. This indicates that HRP-labeled AGG-BNC-ZZ has a higher antibody binding activity than HRP-labeled BNC-ZZ. From the above results, it is shown that both HRP-labeled AGG-BNC-ZZ and HRP-labeled BNC-ZZ have antibody binding activity superior to that of antibodies.

Example 23
An experiment was conducted to evaluate the protein G-derived antibody binding ability of HRP-labeled AGG-BNC-ZZ. In place of the control pig-derived IgG shown in Example 22, BSC-ZZ was considered to be difficult to bind, and mouse-derived IgG ₁ was used for solid phase, and the other experiments were performed. The results are shown in FIG.

HRP-labeled AGG-BNC-ZZ showed high binding reaction to mouse IgG _1. On the other hand, almost no detection was possible with HRP-labeled BNC-ZZ. This is because the antibody binding site derived from protein G of HRP-labeled AGG-BNC-ZZ functions well, and HRP-labeled AGG-BNC-ZZ is high even for antibodies that are difficult to bind at the antibody binding site derived from protein A. It shows that it has a binding force.

Example 24
An application experiment of HRP-labeled AGG-BNC-ZZ to ELISA was performed. GFP protein produced using E. coli was immobilized on an ELISA plate and then blocked using k-Block-e. A rabbit-derived anti-GFP antibody was added to each well at various concentrations shown on the horizontal axis of the graph of FIG. 10, and further 100 ng / mL HRP-labeled AGG-BNC-ZZ [Production Example 6] or SH-HRP-labeled BNC-ZZ [ Production Example 3] was added as a probe for reaction, and after washing, Abs 450 nm was measured in the same manner as in Example 9. Pluronic F-127 was added to the probe so that the final concentration was 0.05%. The results are shown in FIG.

  HRP-labeled AGG-BNC-ZZ, like SH-HRP-labeled BNC-ZZ used as a control, showed a reaction depending on the added antibody concentration, but the former reaction was about ½ compared to the latter. . Considering that the HRP activity of HRP-labeled AGG-BNC-ZZ is 1/3 that of HRP-labeled BNC-ZZ, it was found that HRP-labeled AGG-BNC-ZZ exhibits a reaction higher than that of HRP-labeled BNC-ZZ.

Subsequently, instead of the rabbit-derived anti-GFP antibody, a solid-phase treatment was performed using a mouse-derived anti-HMG1 monoclonal antibody (Cosmobio; IgG ₁ ), and the experiment was performed in the same manner except that. The results are shown in FIG.

  When HRP-labeled AGG-BNC-ZZ was used as a detection probe, a reaction depending on the added antibody concentration was shown, but in the case of HRP-labeled BNC-ZZ, almost no reaction was observed.

The above results indicate that HRP-labeled AGG-BNC-ZZ can be used in an antibody detection type ELISA even in a practical measurement system. Further, HRP-labeled AGG-BNC-ZZ indicates that mouse IgG ₁ that cannot be detected by HRP-labeled BNC-ZZ can also be detected, indicating that it is very useful.

Example 25
An experiment was conducted to examine the antibody binding activity of the HRP-labeled BNC-ZZ / rabbit-derived anti-mouse IgG antibody complex. The ELISA plate was immobilized using control mouse-derived IgG, and then blocked by reacting with k-Block-e for 1 hour. Among the HRP-labeled BNC-ZZ / rabbit-derived anti-mouse IgG antibody complex [Production Example 7] produced using the cross-linker BS ₃ in each well, those prepared with cross-linker BS ₃ concentrations of 200 μM and 1000 μM, Methods having the concentrations of 0, 0.55, 1.65, 4.94, 14.8, 44.4, 133, and 400 ng / mL were added and reacted as probes, and after washing, the same method as in Example 9 Abs 450 nm was measured. Pluronic F-127 was added to the probe so that the final concentration was 0.05%. The results are shown in FIG.

  A reaction depending on the concentration of both complexes used was observed. When the cross-linking agent at the time of preparation of the complex was compared between 200 μM and 1000 μM, the latter showed a slight decrease in binding activity, but both showed a practical level of antibody binding activity.

Example 26
It was confirmed whether anti-OVA mouse IgE and anti-OVA mouse IgG present in anti-OVA mouse antiserum obtained by immunization with ovalbumin (OVA) could be measured practically. OVA was immobilized on an ELISA plate and then blocked using h-Block-e. To this plate, each anti-OVA mouse antiserum diluted 100 times or more was added. As a probe for measuring IgE, a complex prepared by using SH-HRP labeled BNC-ZZ [Production Example 3] and anti-mouse IgE (Nordic immunology Lab) according to the method of [Production Example 7] at a BS ₃ concentration of 1000 μM, and HRP labeling Anti-mouse IgE was used. As a probe for IgG measurement, a mixed complex of SH-HRP-labeled BNC-ZZ [Production Example 3] and rabbit-derived anti-mouse IgG antibody was used. These probes were added to the wells of the plate prepared for each measurement, reacted, washed, and then measured for Abs 450 nm in the same manner as in Example 9. The measurement value was calculated using mean ± std (unit / mL, n = 3) based on the antibody titer of the standard antibody, using a calibration curve prepared using the standard antibody.

  The antibody titer of anti-OVAIgE in mouse serum is 3807 ± 1439 nunit / mL when HRP-labeled BNC-ZZ / anti-mouse IgE complex is used, and 3558 ± 935 nunit / mL when HRP-labeled anti-mouse IgE is used. Yes, both showed similar values.

  The antibody titer of anti-OVA mouse IgG was 4175 ± 8717 μunit / mL. As described above, it was found that the antibody titer measurement of IgE and IgG is sufficiently possible in the measurement system using these complexes and has practical utility.

Example 27
Using the HRP-labeled BNC-SA / rabbit-derived anti-mouse IgG antibody complex prepared in [Production Example 8], an experiment was conducted to confirm the binding reaction to mouse IgG. Control mouse-derived IgG was immobilized on an ELISA plate and then blocked using 1% Block Ace. HRP-labeled BNC-SA / rabbit-derived anti-mouse IgG antibody complex [Production Example 8] as a probe in each well, and NH ₂ -HRP-labeled BNC-ZZ [Production Example 3] and an anti-mouse IgG rabbit antibody (Bethyl) as controls And a probe derived from a rabbit-derived HRP-labeled anti-mouse IgG antibody were added and reacted at concentrations of 0, 0.55, 1.65, 4.94, 14.8, 44.4, 133, and 400 ng / mL, and after washing, Example 9 Abs 450 nm was measured by the same method as described above. Pluronic F-127 was added to the probe so that the final concentration was 0.05%. The result is shown in FIG.

The response increased depending on the concentration of both probes. The HRP-labeled BNC-SA / antibody complex showed about 1/2 of the binding activity as compared with the mixed complex of HRP-labeled BNC-ZZ and antibody in which HRP was labeled through the same NH ₂ group. If the enzymatic activity of the HRP think the former is about 1/3, HRP-labeled BNC-SA / antibody complexes high ability to bind to the antibody from the mixed complex of NH ₂ over HRP-labeled BNC-ZZ and antibody It shows that. On the other hand, when compared with the HRP-labeled antibody, the HRP-labeled BNC-SA / antibody complex showed about twice the reaction. As described above, it has been found that the HRP-labeled BNC-SA / antibody complex has a higher binding ability to the antibody than the HRP-labeled anti-mouse IgG antibody and is useful.

Example 28
In the same manner as in Example 13, the HuH7 cell extract was subjected to Western blotting. The membrane was blocked with 5% skim milk, and the primary antibody used was a mouse-derived anti-vimentin antibody (Progen, 1/1000 dilution). HRP-labeled anti-mouse IgG antibody from rabbit containing 1% skim milk as a probe (Rockland, 1/10000; 2nd Ab in the figure), or HRP-labeled BNC containing 0.1% Pluronic F-127 and 1% skim milk -ZZ [Production Example 3] was used. The results are shown in FIG.

  When detection was performed using a rabbit-derived HRP-labeled anti-mouse IgG antibody as a probe, no signal of the band could be confirmed when the extract was diluted to 1/10. However, when HRP-labeled BNC-ZZ was used, 1 / An equivalent signal was obtained even with an extract diluted to 10. Therefore, it turns out that HRP label | marker BNC-ZZ is effective for highly sensitive detection.

Example 29
Western blotting was performed in the same manner as in Example 28. The anti-vimentin mouse antibody used as the primary antibody was diluted 1/3000 and detected with the HRP-labeled anti-mouse IgG antibody (Rockland, # 611-1302, 1/10000 dilution) as the secondary antibody (Detect- in the figure) 1) After that, it was further dissolved in a solution containing 0.1% Lipidure 802 to detect HRP-labeled BNC-ZZ as an additional probe (Detect-2 in the figure). The results are shown in FIG.

  If the detection sensitivity of the HRP-labeled anti-mouse antibody used as the secondary antibody is poor (Detection-1), the band signal can be strengthened simply by re-detection (Detection-2) using HRP-labeled BNC-ZZ as an additional probe. You can see that it was made. Although no data is shown, the signal hardly increased even when a secondary antibody was added. Therefore, HRP-labeled BNC-ZZ is very useful because it can sensitize a signal only by adding it.

Example 30
Western blotting was performed in the same manner as in Example 28. Anti-vimentin mouse antibody (Progen, 1/2000 dilution), anti-GAPDH rabbit antibody (EPITOMICS, 1/10000 dilution), or both as primary antibody, HRP-labeled anti-mouse IgG antibody (Rockland) as secondary antibody , HRP-labeled anti-rabbit IgG antibody (Santa Cruz), or HRP-labeled BNC-ZZ (HRP-ZZ). Note that HRP-labeled BNC-ZZ was used together with Pluronic F-127 in such an amount that the final concentration was 0.1%. The results are shown in FIG.

  When HRP-labeled BNC-ZZ is used, it can be seen that vimentin and GAPDH can be detected at one time at the position detected by each secondary antibody. It was also shown that HRP-labeled BNC-ZZ can be detected at the same time even if the animal species from which the antibody is derived is different.

Example 31
As a sample, GFP-Histag protein whose concentration was adjusted in a 2-fold dilution series was added to the HuH7 cell extract, and this was subjected to Western blotting in the same manner as in Example 28. HRP diluted with PBS-T containing 0.1% Lipidure 206 after reacting with anti-GAPDH rabbit antibody (EPITOMICS, 1/10000) and anti-GFP rabbit antibody (Rockland, 1/2000) as primary antibodies Simultaneous detection using labeled BNC-ZZ (HRP-ZZ). The results are shown in FIG.

  When HRP-labeled BNC-ZZ was used, it was found that the signal of GFP protein was enhanced according to the added concentration, and was quantitative.

Example 32
Western blotting was performed in the same manner as in Example 28. Anti-vimentin mouse antibody (Progen, 1/2000) and HRP-labeled BNC-ZZ were mixed in equal amounts in advance, and the mixed complex was added to the PVDF membrane to detect by one-step method. Note that TBS-T containing 0.1% Pluronic F-127 was used as the reaction solution of the mixed complex of HRP-labeled BNC-ZZ and antibody. As a control, it was also detected by a two-step operation in which a reaction with an anti-Vimentin mouse antibody was followed by a reaction with an HRP-labeled anti-mouse antibody (Rockland, 1/5000). The results are shown in FIG. In the one-step method, after transfer of the protein to the PVDF membrane, the operation time until detection is 5 minutes for blocking (Q1 in the figure) or 15 minutes (Q2 in the figure), then 5 minutes for the washing to be performed, and then The reaction to be performed (primary antibody + HRP-labeled BNC-ZZ) is 30 minutes, then the washing to be performed is 25 minutes (5 minutes × 5: Q1) or 15 minutes (3 minutes × 5: Q2), and the total time is about 65 minutes On the other hand, in the two-step method using an HRP-labeled anti-mouse antibody (M in the figure), blocking is performed for 60 minutes, then washing is performed for 10 minutes (5 minutes × 2), then primary antibody reaction is performed for 60 minutes, The subsequent washing was 15 minutes (5 minutes × 3), the subsequent secondary antibody reaction was 60 minutes, the subsequent washing was 25 minutes (5 minutes × 5), and the total time was about 230 minutes.

  The conventional two-step method requires a total of 230 minutes until the signal is detected using the protein transfer membrane. However, the one-step method using the HRP-labeled BNC-ZZ (Q1, Q2 in the figure). In the case of), the cleaning time and other times were shortened to 65 minutes, and the same result could be obtained. Therefore, it was found that HRP-labeled BNC-ZZ has utility that enables rapid detection Western blot.

Example 33
Western blotting was performed in the same manner as in Example 28. Anti-p53 rabbit antibody (Santa Cruz, 1/200) as the primary antibody, ALP-labeled anti-rabbit IgG antibody (Sigma, 1/50000) as the secondary antibody, or SH-ALP-labeled BNC-ZZ [Production Example 4] CDP-Star (NEB) was used as a substrate for ALP. The results are shown in FIG.

  When detected with ALP-labeled anti-rabbit IgG antibody and when detected with ALP-labeled BNC-ZZ, almost the same signal was obtained, and ALP-labeled BNC-ZZ is a highly useful probe even in Western blotting. HRP-labeled BNC- It was suggested that various uses shown in the examples of the present application using ZZ are possible.

Example 34
Western blotting was performed in the same manner as in Example 28 except that A431 cell extract was used. After antibody species as the primary antibody reacted with an anti-EGFR antibody is a mouse _{IgG 1 (Cell Singnaling, 1/} 1000 dilution) or anti-p53 rabbit antibody (Santa Cruz, 1/200 dilution), 0.1% The reaction was carried out with HRP-labeled AGG-BNC-ZZ [Production Example 6] or HRP-labeled BNC-ZZ [Production Example 3] diluted with TBS-T containing Pluronic F-127. The results are shown in FIG.

When an anti-EGFR antibody that is mouse IgG ₁ was used, HRP-labeled BNC-ZZ (Z in the figure) could not be detected at all, but HRP-labeled AGG-BNC-ZZ (A in the figure) was used. In some cases, detection was possible. On the other hand, when the anti-p53 antibody, which is a rabbit IgG, was used, detection was possible using either HRP-labeled BNC-ZZ or HRP-labeled AGG-BNC-ZZ.

These results, HRP labeled AGG-BNC-ZZ is also readily bound to the mouse IgG ₁ for having the antibody binding site derived from a protein G, HRP-labeled BNC-ZZ because with only the antibody combining site of protein A from good agreement with that hardly bind to mouse IgG _1, utility of the HRP-labeled AGG-BNC-ZZ was demonstrated.

[Production Example 9: Preparation of HRP-labeled BNC-L]
HRP labeling was performed through the SH group of BNC-L [Production Example 1] using Peroxidase Labeling Kit-SH to obtain HRP-labeled BNC-L.

Example 35
An ELISA plate on which Pre-S2, which is a peptide of the surface antigen of hepatitis B virus, was immobilized was blocked, and various concentrations of anti-Pre-S2 antibody were added to each well. Next, HRP-labeled BNC-L [Production Example 9] was added and reacted, and after washing, Abs 450 nm was measured by the method shown in Example 9 (antigen sandwich ELISA). The result is shown in FIG.

  As a result, it was revealed that HRP-labeled BNC-L can be sufficiently used as a probe for ELISA measurement.

[Production Example 10: SH-HRP-labeled BNC- (sugar chain) -AGG]
BNC-L [Production Example 1] was oxidized with NaIO ₄ to form an aldehyde group on the sugar residue in the sugar chain added to BNC-L. Subsequently, it was reacted with AGG protein to bind the aldehyde group and the lysine residue of the AGG peptide. NaBH ₄ solution was added to the reaction product and subjected to gel filtration to obtain BNC-AGG. Furthermore, HRP labeling was performed via the SH group of the obtained BNC-AGG using Peroxidase Labeling Kit-SH. That is, AGG is bound to the obtained BNC via its sugar chain, and further, HRP is bound via its SH group (hereinafter referred to as SH-HRP-labeled BNC- (sugar chain) -AGG). Sometimes called).

Example 36
The ELISA plate solid-phased with various concentrations of rabbit IgG was blocked, and SH-HRP-labeled BNC- (sugar chain) -AGG [Production Example 10] was added to each well for reaction. After washing, Example 9 Abs 450 nm was measured by the method shown. The results are shown in FIG.

  The Abs 450 nm value by SH-HRP-labeled BNC- (sugar chain) -AGG was lower than that of SH-HRP-labeled BNC-ZZ [Production Example 4] used as a control, but was found to have sufficient detection ability. .

[Production Example 11: Preparation of HRP-labeled HVJ-E]
Using Peroxidase Labeling Kit-SH, HRP can be transmitted via SH group at the cysteine residue of protein present on the surface of HVJ-E (Genome One, Ishihara Sangyo), a virus-like particle containing Sendai virus envelope protein. Labeling was performed to obtain HRP-labeled HVJ-E.

Example 37
The HRP activity [Production Example 11] of HRP-labeled HVJ-E was measured by the same method as in Example 1. The result was 0.05 U / μg.

  Virus-like particles having a transmembrane protein often have SH groups in or near the transmembrane region of the protein. This example shows that labeling via SH groups is possible even in HVJ-E, and that SH groups present in virus-like particles are useful as labeling targets.

Claims (6)

  A virus-like particle for immunological measurement containing a protein having self-organization ability, wherein the protein having self-assembly ability is HBsAg protein, and at least one cysteine residue of the protein having self-assembly ability A virus-like particle, which is modified by an enzyme via the thiol group.   The virus-like particle according to claim 1, wherein the protein having the ability to self-assemble comprises the amino acid sequence shown in SEQ ID NO: 1.   The virus-like particle according to claim 1 or 2, wherein the protein having the ability to self-assemble has an antibody binding domain.   The virus-like particle according to any one of claims 1 to 3, wherein the enzyme is any selected from the group consisting of peroxidase and alkaline phosphatase. The virus-like protein according to claim 3 , wherein the antibody-binding domain is at least one selected from the group consisting of an antibody-binding domain in protein A, an antibody-binding domain in protein G, and an antibody-binding domain in protein L. particle. The virus-like particle according to claim 3 , wherein the antibody-binding domain has an amino acid sequence represented by any one of SEQ ID NOs: 3 to 5.